DTU Nanotech, Department of Micro- and Nanotechnology, Technical University of Denmark, Building 423, 2800 Lyngby, Denmark.
ACS Nano. 2011 Jul 26;5(7):5864-73. doi: 10.1021/nn201643f. Epub 2011 Jul 6.
Particle-based nanosensors have over the past decade been designed for optical fluorescent-based ratiometric measurements of pH in living cells. However, quantitative and time-resolved intracellular measurements of pH in endosomes and lysosomes using particle nanosensors are challenging, and there is a need to improve measurement methodology. In the present paper, we have successfully carried out time-resolved pH measurements in endosomes and lyosomes in living cells using nanoparticle sensors and show the importance of sensor choice for successful quantification. We have studied two nanoparticle-based sensor systems that are internalized by endocytosis and elucidated important factors in nanosensor design that should be considered in future development of new sensors. From our experiments it is clear that it is highly important to use sensors that have a broad measurement range, as erroneous quantification of pH is an unfortunate result when measuring pH too close to the limit of the sensitive range of the sensors. Triple-labeled nanosensors with a pH measurement range of 3.2-7.0, which was synthesized by adding two pH-sensitive fluorophores with different pK(a) to each sensor, seem to be a solution to some of the earlier problems found when measuring pH in the endosome-lysosome pathway.
基于粒子的纳米传感器在过去十年中被设计用于基于光学荧光的活细胞内 pH 值的相对测量。然而，使用粒子纳米传感器对内涵体和溶酶体中的 pH 值进行定量和时间分辨的细胞内测量具有挑战性，需要改进测量方法。在本文中，我们成功地使用纳米颗粒传感器在活细胞的内涵体和溶酶体中进行了时间分辨的 pH 值测量，并展示了传感器选择对于成功定量的重要性。我们研究了两种通过内吞作用内化的基于纳米粒子的传感器系统，并阐明了纳米传感器设计中的重要因素，这些因素应在未来新型传感器的开发中加以考虑。从我们的实验中可以清楚地看出，使用具有较宽测量范围的传感器非常重要，因为当测量 pH 值接近传感器敏感范围的极限时，错误的 pH 值定量是一个不幸的结果。通过向每个传感器添加两个具有不同 pK(a) 的 pH 敏感荧光团来合成具有 3.2-7.0 pH 测量范围的三标记纳米传感器，似乎是解决在内涵体-溶酶体途径中测量 pH 值时早期发现的一些问题的一种方法。
